Alternative splicing is an important cellular process that contributes to proteome diversity. It is estimated that greater than 95% of all genes undergo alternative splicing. These alternative splicing events are often spatially and temporally regulated and generated in response to external stimuli. In general, the regulation of alternative splicing is achieved through complex interplay between cis-regulatory elements within the pre-mRNA and the trans protein factors that bind them. Trans-binding protein factors belong to two general classes: serine-arginine rich (SR) proteins and heterogenous ribonucleoproteins (hnRNPs), whose canonical roles are to either promote or repress the inclusion of an exon in the nascent pre-mRNA transcript, respectively. The balance in the levels of these factors and their binding to specific sites on the pre-mRNA is key toward influencing the decisions of the spliceosome, thereby enabling splicing regulation. SRSF1, formerly SF2/ASF, is one such member of the serine-arginine rich family of SR proteins. In addition to its role in alternative splicing, SRSF1 is required to mediate canonical splicing events including 5′ splice site selection and lariat formation of the major spliceosome. Krainer et al., Cell, 62, 35-42 (1990); Li, X. and Manley, J. L. Cell, 122, 365-378 (2005). SRSF1 is an important proto-oncogene due to its role in the alternative splicing regulation of several cancer-associated genes. Karni, et al., Nat Struct Mol Biol, 14, 185-193 (2007).
Murine Double Minute 2 (MDM2) is an E3 ubiquitin ligase and negative regulator of the tumor suppressor protein p53. Under normal conditions, MDM2 is constitutively spliced to generate a full-length protein, which self-dimerizes and promotes the proteasome-mediated degradation of p53. Fang et al., J Biol Chem, 275, 8945-8951 (2000). However, under stress MDM2 undergoes alternative splicing, generating splice variants that are unable to bind and regulate p53. Chandler et al., Cancer Res, 66, 9502-9508. Subsequently, p53 becomes upregulated and activates downstream targets involved in apoptosis and cell cycle arrest. Jacob et al., PLoS One, 9, e104444 (2014). MDM2-ALT1, which consists of only the two terminal coding exons 3 and 12, is the most frequently observed of these splice isoforms. Despite studies characterizing MDM2-ALT1 as a dominant negative regulator of full-length MDM2 and its pervasiveness in various cancers (Sigalas et al., Nat Med, 2, 912-917 (1996); Yu et al., Cancer, 118, 1110-1118 (2012)), there is very little known about the regulation of MDM2 alternative splicing in cancer and under stress.
It is known that MDM2 splicing occurs in cells in response to UV irradiation and cisplatinum treatment in a manner independent of the p53, ATM and ATR status of these cells. Additionally, co-transcriptional regulation of MDM2 splicing has been demonstrated in response to camptothecin. In this case, the disruption of the interaction between the Ewing's Sarcoma Protein (EWS), which interacts with RNA Polymerase II (Pol II) and the spliceosome-associated factor Y-box-binding Protein 1 (YB-1) upon camptothecin treatment results in the uncoupling of transcription and splicing and ultimately the alternative splicing of MDM2. Dutertre et al., Nat Struct Mol Biol, 17, 1358-1366 (2010). However, MDM2 alternative splicing can also occur independently of transcription as demonstrated by in vitro cell-free splicing systems that utilize nuclear extracts from normal and UV or cisplatinum-treated cells. Singh et al., Exp Cell Res, 315, 3419-3432 (2009). Using such in vitro splicing assays in conjunction with a stress-responsive MDM2 minigene, the inventors previously identified conserved positive sequences within intron 11 of MDM2 and binding factors such as FUBP1 that are important for its efficient splicing. Jacob et al., J Biol Chem, 289, 17350-17364 (2014).